Fixing Cells for Flow Cytometry

Developed by: Justin Meyers

Sample: Cell line or primary cells


  • Cells stained with antibody
  • BD Cytofix (BD 554655)
  • D-PBS
  • FBS


Procedure for fixing cells with BD Cytofix™

  1. Pellet 106 cells by centrifugation (250 - 300 x g) and carefully remove supernatant
  2. Make up 1X of fixation buffer by adding 5 ml of Cytofix (BD554655) to 10 ml of DPBS
  3. Add either 300μl (for microwell plates) or 500 μl (for tubes) aliquots of 1X fixation buffer to each cell pellet and resuspend the cells by either pipetting or vortexing.
  4. Incubate the cells with fixation buffer for 15 to 30 min at 4°C. (Cell aggregation can be avoided by vortexing prior to the addition of the fixation buffer.)
  5. Fixed cells should be washed and suspended in a buffer that contains protein. (DPBS + 5% FBS) for longer term storage. They can be left in the fixative for up to two days.

Store the fixed cells at 4°C (protected from light).

It is recommended that fixed cell samples be read as soon as possible, i.e., within one week.


Jill Hutchcroft
Flow Cytometry Facility Director
Phone: (765) 586-1130